This invention relates, in general, to analytical systems for separating and detecting polyhalogenated diaromatic hydrocarbons.
Polyhalogenated diaromatic hydrocarbons (PHDHs) are a diverse group of widespread environmental contaminants that include polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs), polychlorinated dibenzo-p-dioxins (PCDDs), polybrominated biphenyls (PBBs), polybrominated dibenzofurans (PBDFs), polybrominated dibenzo-p-dioxins (PBDDs), and other subclasses of PHDHs. See, e.g., Giesy et al, Chapter 9, pp. 249-307, Dioxins and Health (ed., A. Schechter, Plenum Press, New York, 1994). Of the PHDHs, the chlorinated compounds or polychlorinated diaromatic hydrocarbons (PCDHs) have been most widely studied. However, the environmental and health impacts of other PHDH compounds, such as polybrominated diaromatic hydrocarbons and mixed brominated/chlorinated diaromatic hydrocarbons are increasingly being recognized.
Many PHDHs are lipophilic and resistant to degradation, and have been detected in soil, sediment, and water. Moreover, many PHDHs have been found to concentrate and accordingly amplify their effect in the food chain. See, e.g., Giesy et al. (1994a), supra; Tanabe et al., Environmental Pollution 47, 147-163 (1987); Giesy et al, Environmental Science and Technology, 28, 128A-135A (1994b); Webster et al., Chapter 1, pp. 1-6, Dioxins and Health (ed., A. Schechter, Plenum Press, New York, 1994). Exposure to and bioaccumulation of PHDHs have been observed to produce a variety of deleterious species- and tissue-specific effects, including tumor promotion, birth defects, hepatotoxicity, immunotoxicity, dermal toxicity, alterations in endocrine homeostasis, undesirable induction of numerous enzymes (including cytochrome P450 1A1), and death. See e.g., Giesy et al., 1994b, supra, Poland et al., Ann. Rev. Pharmacol. Toxicol. 22, 517-542 (1982); Safe, Critical Reviews in Toxicology 24, 87-149 (1994); DeVito and Birnbaum, in Dioxins and Health, pp. 139-162 (ed., A. Schechter, Plenum Press, New York, 1994)
Known techniques for detection and quantification of PHDHs generally involve costly and time-consuming traditional instrumental analysis methods, such as gas chromatography separation (GC) and mass spectrometry (MS). These methods generally involve extensive sample processing before quantification and analysis. See, e.g., United States Environmental Protection Agency (EPA) Method 1613 (40 C.F.R. Part 136) and EPO Method 8290 (40 C.F.R. Part 261). High resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) processing generally requires an extraction of the sample to be analyzed, then treatment of the resulting extract with acid and base compounds, followed by sequential separations (usually by chromatography techniques) on silica gel columns impregnated with, e.g., sulfuric acid, neutral alumina, silica gel and activated carbon; This multiple-step processing of sample extracts usually yields an extract sufficiently free of interfering compounds to yield a congener-specific quantification of many, but not all, toxic PHDHs using HRGC/HRMS detection.
A significant disadvantage of the HRGC/HRMS technique is that, in general, only polychlorinated compounds (e.g., PCDHs) can be quantified by the method. For example, analytical techniques for detecting/quantifying polybrominated compounds and mixed brominated/chlorinated compounds are currently very costly and their standards not well-established. Unfortunately, it is now thought that polybrominated or mixed polybrominated/polychlorinated compounds are as toxic or even more toxic than corresponding chlorinated isomers. There accordingly remains a need for systems and methods that can determine and quantify toxic compounds not detectable by the HRGC/HRMS methods, such as toxic polybrominated or mixed polybrominated/polychlorinated diaromatic hydrocarbons.
Moreover, although HRGC/HRMS analysis allows detection and quantification of mixtures of known PCDH isomers and congeners, the method generally does not provide any information about the biological and/or toxicological effects of these complex mixtures. These mixtures, under certain environmental conditions, may theoretically contain up to or even more than 210 different PCBs, 135 different PCDFs, and 75 different PCDD isomers and congeners, each of which have vastly different chemical, physical, and toxicological properties. See Safe, 1994, supra, and Safe, Critical Reviews in Toxicology 21, 51-88 (1990).
Accordingly, accurate prediction of the biological/toxic activity of complex PCDH mixtures using HRGC/HRMS is difficult. Consequently, the toxicological potency of a complex mixture of PCDH chemicals is generally assessed by the toxic equivalent factor (TEF) approach, in which the concentration of individual compounds present in the PCDH mixture are multiplied by their specific TEF, and the sum of the values expressed as toxic equivalents (TEQs). See Safe, 1994, supra; Safe, 1990, supra; and Ahlborg et al., European J. of Pharmacol and Environmental Toxicology 228, 179-199 (1992).
It is generally thought that many effects of PHDDs, PHDFs, and dioxin-like PHBs proceed through the action of the aryl hydrocarbon receptor (AhR), a cytosolic protein that binds these compounds with high affinity (Safe 1990, supra; S. Safe, Environ. Health Perspect. 100, 259-268 (1992); Safe 1994, supra; L. S. Birnbaum, Environ. Health Perspect. 102, Suppl. 9, 157-167 (1994); M. S. Denison and S. Heath-Pagliuso, Bulletin of Environmental Contamination and Toxicology 61, 557-568 (1998); M. S. Denison et al., xe2x80x9cThe Ah receptor signal transduction pathway,xe2x80x9d in Denison, M. S., Helferich, (Eds.), Xenobiotics, receptors and gene expression (Taylor and Francis, Philadelphia, pp. 3-33, 1998a). The occurrence of this common mechanism supports the continued use of the TEF concept.
PCDDs and PCDFs with chlorine substitutions in the 2-,3-,7-, and 8-positions exhibit the highest affinity binding to the AhR and the strongest toxic effects, with increasing chlorination generally reducing potency. The toxic potencies of the different congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), the most toxic dioxin, are indicated by their corresponding toxic equivalency factors (TEFs). Over the past decade, a number of different TEF schemes have been proposed. See e.g., U. G. Ahlborg, Chemosphere 28, 1049-1067 (1994); M. Van den Berg et al., Environ. Health Perspect. 103, 775-792 (1998); S. Safe, Teratogenesis, Carcinogenesis and Mutagenesis 17, 285-304 (1997-98). The most recent consensus TEFs for humans, fish and wildlife risk assessment were derived at a WHO/IPCS (World Health Organization/International Programme on Chemical Safety) meeting held in Stockholm, Sweden on Jun. 15-18, 1997. See Van den Berg et al., 1998, supra, and M. Van den Berg, Food Add. Contam. 17, 347-358 (2000). TEFs have been assigned to seven PCDDs, ten PCDFs and twelve PCBs, and range from 0.00001 to 1, reflecting a pronounced variability in toxicity.
Hazard and risk assessments of chemicals carried out by regulatory agencies have primarily addressed the toxicities of individual compounds, whereas humans and wildlife are exposed to complex mixtures of toxic compounds (Safe, 1998, supra). In addition to PCDDs, PCDFs, and dioxin-like PCBs, a number of other compounds exert AhR-agonist activity. In 1998, the international WHO committee agreed that other halogenated chemicals meet the criteria for inclusion in the TEF concept, but maintained that insufficient environmental and toxicological data were available to establish TEF values for these compounds (Van den Berg et al., 1998, supra). A few examples of these compounds, detailed further below, are brominated and mixed chloro/bromo-substituted analogues of PCDDs, PCDFs and PCBs, halogenated naphthalenes, halogenated diphenyl ethers, halogenated azo- and azoxybenzenes and polycyclic aromatic hydrocarbons (PAHs). See, e.g., G. Sundstrom et al., Chemosphere 15, 2105-2107 1986; Birnbaum, 1994, supra; Denison and Heath-Pagliuso, 1998, supra; and Denison et al., 1998b, supra.
Cell-based bioassays have been combined with extraction technology and acid silica gel column chromatography in order to improve the analysis of the specificity of PCDHs contaminating environmental samples. For example, in one known methodology, the correlation of the determination of specific content of PCDHs with the results of traditional HRGS/HRMS techniques was about 0.71. However, this methodology and others have certain disadvantages, including the inability to quantify the content of PCBs relative to the content of the dioxin/furan class of PCDHs in a mixture. See J. M. Aarts et al., Organohalogen Compounds 27, 285-289 (1996).
Cell bioassay systems have been developed to directly quantify TEQ activity based upon a concentration-specific expression of the toxicity of certain PCDHs. U.S. Pat. No. 5,854,010 to Denison et al. (hereinafter the xe2x80x9c+010xe2x80x9d patent, which patent is incorporated by reference herein in its entirety) describes a detection system based on a novel recombinant cell line that responds to certain polyaromatic hydrocarbons by expressing the reporter compound luciferase. See also P. M. Garrison, et al., Fundam. Appl. Toxicol. 30, 194-203 (1996). In other words, the bioassay system of the ""010 patent (also and hereinafter interchangeably referred to as the CALUX(copyright) system) responds to the presence of certain polyaromatic hydrocarbons by luminescence, which can be quantified and ultimately accurately correlated with the amount of the polyaromatic hydrocarbons present in a sample.
Despite having advantages over previous methods of polyaromatic hydrocarbon quantification, the system described by the ""010 patent still requires time-consuming sample processing methods in order to provide results that reliably correlate with the results obtained with isomer-specific detection by HRGC/HRMS methods generally used for regulatory purposes (i.e., EPA Methods 8290 and 1613). Accordingly, a need still exists for reliable and convenient sample processing techniques for separating individual types of PHDH compounds (e.g., PCDH and PBDH compounds) for quantification and analysis. Additionally, sample processing and evaluation techniques are needed for determining the presence and toxicity of a broad range of toxic polyhalogenated compounds, including polybrominated and mixed brominated/chlorinated compounds.
The present inventors have discovered a rapid, relatively inexpensive methodology to quantify TEQ contributed by polyhalogenated biphenyls (e.g., PCBs and PBBs), and/or dioxins and/or furans of the PHDH class of compounds. In one embodiment, the current invention describes separation methods useful for providing samples for the isolation and specific detection of 2,3,7,8-tetrachlorodibeno-para-dioxin and related toxic congeners from the family of polychlorinated diaromatic hydrocarbons (PCDH). The method allows for the quantification of specific toxic compounds of the PHDH family of compounds. The inventive method also allows for accurate resolution of the toxicity due to polyhalogenated, and particularly, polychlorinated dibenzodioxins and furans respective to the toxicity contributed by the polyhalogenated, and particularly polychlorinated biphenyls.
Accordingly, the present invention provides sample processing methods for the quantitative analysis of certain polyaromatic hydrocarbons (and particularly, polyhalogenated diaromatic hydrocarbons). One embodiment of the method comprises separating components of a polyaromatic hydrocarbon mixture from a sample and determining the amount of both total polyaromatic hydrocarbons and specific families of polyaromatic hydrocarbons in the sample.
In certain embodiments, the present method may be used in conjunction with known methods for quantifying families of polyaromatic hydrocarbon compounds within complex mixtures. Such quantifying methods include but are not limited recombinant bioassay systems for testing for polyhalogenated diaromatic hydrocarbons, and, in the case of polychlorinated diaromatic hydrocarbons, traditional chemical analysis of gas chromatography with electron capture detection (GC/ECD) and analysis with high resolution gas chromatography high resolution mass spectrometry (HRGC/HRMS).
In a preferred embodiment, the quantification method is the bioassay system known as the CALUX(copyright) system described in U.S. Pat. No. 5,854,010 (incorporated by reference herein in its entirety), which utilizes using a recombinant cell line deposited at the American Type Culture Collection under accession number CRL-12286 (also known and referred to herein interchangeably as the xe2x80x9cmouse H1L1.1 cell linexe2x80x9d).
The present invention method advantageously allows for the preparation of a sample so that multiple determinations can be made of the content of the polyaromatic hydrocarbons present in the sample. Certain embodiments of the invention allow for individual quantification of all polyaromatic hydrocarbons that are present in the sample. Additional aspects of the invention provide for the removal of certain chemical substances that are not of the class of compounds that are PHDHs.
Using the inventive methods described herein, PHDHs, and particularly, PCDHs can be quantified by adsorption to an affinity matrix as described herein. These methods may optionally measure/quantify the amount of compounds in a sample that belong to the group of compounds known as planar polychlorinated biphenyls (PCBs), as well as the amount of compounds in a sample that belong to the group comprising polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). These methods may optionally measure/quantify the amount of compounds in a sample that belong to the group of compounds known as planar polybrominated biphenyls (PBBs), as well as the amount of compounds in the sample that belong to the group comprising polybrominated dibenzo-para-dioxins (PBDDs) and polybrominated dibenzofurans (PBDFs). In even other alternative embodiments, these methods may measure/quantify the amount of compounds in a sample that belong to the groups of compounds that are planar mixed polybrominated/polychlorinated biphenyls, as well as the amount of compounds in the sample that belong to the groups comprising mixed polybrominated/polychlorinated dibenzo-para-dioxins and mixed polybrominated/polychlorinated dibenzofurans.
The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.